Although the science of enzymes is almost 100 years old and the key roles that enzymes play in many biochemical reactions were understood 40 years ago, the applications of enzymology to clinical diagnosis are much more recent. As recently as 20 years ago these applications accounted for only about 3-5% of the total number of tests performed in the clinical laboratory, and only four or five enzymes were routinely assayed.
Today, in contrast, enzyme assays may account for as much as 25% of the total work load in larger hospital laboratories, and as many as 20-25 enzymes may be routinely assayed. Detailed procedures for these routine assays may be found in any standard textbook on clinical or diagnostic chemistry.
In an enzyme assay, enzyme concentration in a body fluid, such as serum, urine and cerebrospinal fluid, is determined to be either within a normal range or outside of the normal range. In some assays, an abnormal enzyme concentration is the result of a bacterial or viral infection, and assay for a specific enzyme is diagnostic for a specific pathogen. In other cases, mere detection of an enzyme not normally present in a fluid, such as serum, may be indicative of tissue or organ damage. For example, detection in plasma of alcohol dehydrogenase, which is liver specific, and acid phosphatase, which is prostate specific, pinpoints tissue damage in these organs. Measurement of alkaline phosphatase activity has importance in diagnosing hepatobiliary and bone disorders.
Enzyme assays depend on enzyme catalysis of conversion of a substrate to a product which may be detected or measured. If the concentration of the substrate in an enzyme reaction is gradually increased, keeping all other factors constant, the rate of reaction will increase with increasing substrate concentration until a maximum value is reached. Any further increase in substrate concentration will elicit no further increase in reaction rate. Thus, when the substrate is present in sufficient excess, the rate of reaction depends only on enzyme concentration.
Since enzymes are true catalysts and are not changed during the reaction, the rate of an enzyme reaction, in the presence of sufficient substrate, is constant with time and is dependent only on the concentration of enzyme in the assay system. If the enzyme to be analyzed is present in low concentration, the reaction rate will be very low, and a long period of time may be required in order for sufficient product for detection to form. This is a severe disadvantage in those cases where speed of detection is of the essence, as, for example, in determining the presence of a pathogen in blood or urine.
Another limitation in enzyme analysis is the frequent presence in clinical samples of substances which decrease assay sensitivity by interfering with enzyme activity. These substances, generally referred to as interferences, are particularly troublesome in analysis of enzymes in serum and urine samples. Interferences have conventionally been dealt with by diluting the sample to the point where the interference no longer occurs. This dilution, however, may reduce the enzyme concentration in the sample to the point where it is no longer detectable.
Assay sensitivity in immunoassay has been increased by various amplification procedures. In cascade amplification, the number of detectable (generally colored) molecules is increased by use of two or more enzymes or enzyme derivatives. U.S. Pat. No. 4,463,090 to Harris discloses a cascade amplification immunoassay in which a large molecule activator, such as an enzyme or a proenzyme coupled to a ligand, activates a second enzyme which reacts with a substrate to product a detectable signal or in turn activates a third enzyme.
U.S. Pat. No. 4,446,231 to Self discloses a cycling amplification enzyme immunoassay which includes primary and secondary enzyme systems and a modulator for the second enzyme system. The primary system includes a first enzyme coupled to a ligand. In a first embodiment of the Self invention, the first enzyme system acts on a modulator precursor to liberate a modulator. The modulator is a cofactor of the secondary enzyme which activates the second enzyme system to catalyze the reaction of a substrate to a detectable product. During the reaction, the modulator is converted to an inactive form, and cycling is accomplished by a third enzyme which reactivates the modulator. In a second embodiment the modulator is an inhibitor of the secondary system, and is removed by the primary enzyme system whereby the secondary system is activated to act on the substrate and thereby produce the detectable product. Column 37 of Self discloses use of the patented method for detection of an enzyme.
Boguslaski et al., U.S. Pat. No. 4,492,751 teaches a cycling system in which an enzyme substrate or coenzyme in conjugated to one member of the specifically binding pair.
A variety of molecules has been shown to cause specific inactivation of a target enzyme. A subset of inhibitors, termed mechanism-based inhibitors, are substrates for enzymes which react with an enzyme to form a covalent bond. Mechanism-based inhibitors have been reviewed by Walsh (Tetrahedron 38, 871 (1982)). Another subset of inhibitors includes molecules which act as stable transition-state analogs. Gelb et al. have disclosed some fluoroketones as transition-state inhibitors of hydrolytic enzymes in Biochemistry 24, 1813 (1985).
Enzyme assays are valuable additions to the list of analytical techniques; however, they are subject to the above-described limitations. It is toward improved enzyme assays which solve these problems that this invention is directed.